Excellent preservation techniques for biological cells or tissues are desired in various industrial fields. For example, in the bovine embryo transfer technology, embryos are transferred in consideration of the estrus cycle of a recipient cow. In order to synchronize the transfer of embryos with the estrus cycle, embryos are cryopreserved in advance and are thawed and transferred in time with the estrus cycle. In the human fertility treatment, eggs or ovaries are harvested from a woman's body and cryopreserved until an appropriate timing for transplantation, and the cryopreserved eggs or ovaries are thawed before the use in transplantation.
In general, cells or tissues harvested from living bodies gradually become inactive even in a culture medium, and hence long-term culture of cells or tissues in vitro is undesirable. For this reason, techniques for long-term preservation of cells or tissues without the loss of biological activity are essential. Excellent preservation techniques enable more accurate analysis of cells or tissues harvested. Such excellent preservation techniques also enable transplantation of cells or tissues with their biological activity kept at a higher level, thus likely resulting in an improvement in the engraftment rate. The techniques also enable in-advance production and preservation of artificial tissues for transplantation, such as skins cultured in vitro and what they call cell sheets formed in vitro, and storage thereof until needed. Therefore, such excellent preservation techniques are expected to bring great advantages not only in the industrial fields but also in the medical science fields.
One of known methods for preserving cells or tissues is slow freezing, for example. In this method, cells or tissues are immersed in a preservation solution prepared by adding a cryoprotectant to a physiological solution such as phosphate buffered saline. Examples of the cryoprotectant include compounds such as glycerol and ethylene glycol. The cells or tissues immersed in the preservation solution are cooled down to −30° C. to −35° C. at a relatively slow cooling rate (for example, 0.3° C. to 0.5° C./min), and thereby the solution inside and outside the cells or tissues are sufficiently cooled and become viscous. Further cooling down the cells or tissues in such a state in the preservation solution to the temperature of liquid nitrogen (−196° C.) allows a slight amount of the solution both inside and outside (surrounding) the cells or tissues to become a solid while the amorphous state thereof is maintained, that is, to vitrify. The vitrification (i.e., solidification) of the solution inside and outside the cells or tissues substantially immobilizes the molecules. Thus, the vitrified cells or tissues can be semipermanently preserved in liquid nitrogen.
However, since the slow freezing requires relatively slow-rate cooling, the procedure of cryopreservation takes a long time. Further, this technique disadvantageously needs the use of a temperature-controlling device or jig. In addition, the slow freezing cannot avoid formation of ice crystals in the preservation solution outside the cells or tissues, which may cause physical damage to the cells or tissues.
One proposed solution to the problems of the slow freezing is vitrification cryopreservation. The vitrification cryopreservation is a technique using a principle that addition of a large amount of a cryoprotectant, such as glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO), to water decreases the freezing point of water, thereby restraining formation of ice crystals at sub-zero temperatures. When quickly cooled in liquid nitrogen, such an aqueous solution can solidify without formation of ice crystals. This solidification is called vitrification freezing. The aqueous solution containing a large amount of a cryoprotectant is called a vitrification solution.
The specific procedure of the vitrification cryopreservation is to immerse cells or tissues in a vitrification solution and to cool them at the temperature of liquid nitrogen (−196° C.). Since the vitrification is such a simple and quick process, it advantageously does not require a long-term procedure of cryopreservation or the use of any temperature-controlling device or jig.
The vitrification cryopreservation does not cause formation of ice crystals either inside or outside the cells, and thus can avoid physical damage (freezing damage) to the cells at the time of freezing and thawing. However, a high-concentration cryoprotectant contained in the vitrification solution is chemically toxic. Thus, the volume of the vitrification solution around cells or tissues used in cryopreservation of the cells or tissues is preferably as small as possible. Further, the duration of exposure of the cells to the vitrification solution, that is, the time until freezing, is preferably short. In addition, the vitrification solution needs to be diluted immediately after thawing.
Various examples of the vitrification-based cryopreservation of cells or tissues have been reported using various methods and various cells or tissues. For example, Patent Literature 1 discloses that application of the vitrification cryopreservation to reproductive or somatic cells of animal or human origin is very useful in terms of the cell viability after cryopreservation and thawing.
The vitrification cryopreservation is a technique which has been developed mainly using human reproductive cells. More recently, its application to iPS or ES cells has also been widely examined. Non-Patent Literature 1 discloses the effectiveness of the vitrification cryopreservation in preservation of Drosophila embryos. Patent Literature 2 discloses the effectiveness of the vitrification cryopreservation in preservation of plant culture cells and tissues. As mentioned here, the vitrification is known to be useful for preservation of a wide range and different kinds of cells and tissues.
With regard to devices and procedures for more efficient vitrification cryopreservation, Patent Literature 3, for example, reports an attempt to improve the recovery rate of cryopreserved eggs or embryos by vitrifying them in a straw filled with a vitrification solution, and then bringing them into contact with a diluent immediately in thawing.
Patent Literature 4 proposes a cryopreservation method with excellent viability including depositing eggs or embryos together with a vitrification solution on a removing material for vitrification preservation and removing an excess vitrification solution surrounding the eggs or embryos by downward suction. Examples of the removing material for vitrification cryopreservation disclosed include wire mesh and perforated films made of natural substance, such as paper, or synthetic resin.
Patent Literature 5 proposes a cryopreservation method with excellent viability including absorbing an excess vitrification solution surrounding eggs or embryos with an absorber such as filter paper.
Patent Literature 6 and Patent Literature 7 propose a cryopreservation method, what is called the Cryotop method, used in the field of human fertility treatment. This method uses a tool for cryopreservation of eggs including a flexible, clear and colorless film strip as an egg-holding strip, and includes depositing eggs or embryos together with a very small amount of a vitrification solution on the film under a microscope.
Patent Literature 8 proposes a method of producing frozen cell sheets by the vitrification including placing a cell sheet together with a transfer-support film (Cell Shifter, a sheet mainly made of cellulose, available from CellSeed Inc., or a PVDF film) on a pedestal made of, for example, a wire mesh, absorbing or discharging an excess vitrification solution, and then cryopreserving the cell sheet.